Assessment performed on Friday, May 26, 2017, at Vancouver General Hospital, BC, Canada

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1 Assessors report for ciqc Run 68: p53 Assessors: B Gilks, J Garratt, J Won (recorder) Assessment performed on Friday, May 26, 2017, at Vancouver General Hospital, BC, Canada Overview Interpretation of p53 is tripartite, with either complete absence of staining or strong nuclear staining in at least 80% of cells having evidence of p53 genetic abnormality (so called all or nothing staining pattern). Normal p53 wildtype staining is of variable intensity with weak to moderately intense staining in 1-80% of cells. In general, the higher the proliferation index, the greater the p53 staining in tumours/tissues with wildtype p53 (for example, basal keratinocytes of normal skin show variable p53 positivity while the mitotically inactive superficial keratinocytes are negative). Unlike previous ciqc p53 challenges (Run 42 and 54), self-assessments were done much more consistently according to the all or nothing scoring system described above. Large portions of Cores 6 and 23 were comprised of lymphocytes that were mis-interpreted during selfassessments as positive tumour cell staining. As noted previously, Core 6 is an unusual tumour, in that it has a splicing mutation that should theoretically result in complete absence of p53 protein but many labs show convincing wild type pattern. This case should be disregarded when optimizing the assay. Consistent with Run 42 and 54 observations, some labs continued to have staining that was sufficiently weak as to make it difficult or impossible to distinguish between low-level wildtype and complete absence of p53 expression. Conversely, some labs had significant overstaining issues that made interpretation challenging and led to several incorrect scores. Figure 1. Representative images of staining in Core 23 (an abnormal "nothing"/absent expression case) from select labs with noted staining issues.

2 Participant-specific feedback for p53 IHC is summarized below: Lab ID IHC Status* Assessor comments 101 Optimal 102 Optimal 103 Optimal Slightly weaker staining compared to other labs 104 Failed Overstained making interpretation difficult and very strong counterstain 106 Optimal 107 Optimal 110 Failed Overstained making interpretation difficult 111 Optimal 112 Optimal Notably nice staining No slide 115 Optimal 120 Optimal 123 Optimal Slightly more intense staining compared to other labs 124 Adequate Intense staining; many self-assessment scores altered during ciqc assessment (potential interpretive issue?) 125 Optimal Optimal Slightly weaker staining compared to other labs Optimal 132 Optimal Slightly weaker staining compared to other labs 133 Optimal 138 Failed Extremely weak staining; internal controls generally very faint 141 Failed Overstained making interpretation difficult 144 Optimal 145 Optimal 147 Optimal Generally more intense staining compared to other labs 148 Optimal 149 Sub-optimal Borderline overstained 150 Sub-optimal Very weak staining; internal controls generally faint; very faint counterstain 155 Optimal 159 Optimal Very strong counterstain 160 Optimal 168 Optimal 176 Optimal 183 Failed Extremely weak staining that is difficult to distinguish from counterstain 186 Optimal 202 Adequate Weak staining leading to more "U" due to lack of internal control 209 Optimal No slide 228 Optimal 231 Optimal *Based on assessment team consensus In the Garrattograms in Figure 2, the p53 abnormal results from ciqc assessment were colour coded with pink for "nothing"/absent expression and red for "all"/strong positive staining. Uninterpretable results were also color coded with yellow for complete absence of staining in both tumour cells and normal internal control cells (i.e. lymphocytes or stromal cells) and blue for cases where the core either came off the slide or there was no tumour in the tissue core. Supplementary Table 1 summarizing reported staining protocol details and Supplementary Table 2 summarizing sensitivity, specificity, and Cohen s kappa for each participating laboratory can be found at the end of this document. Quality control methodologies of immunohistochemical assessment are evolving, and numeric results should be interpreted with caution. Your regular participation in ciqc is greatly appreciated and we look forward to working with you and the Canadian Association of Pathologists Association Canadienne des Pathologistes in the future as we continue to improve our external quality assurance services.

3 Figure 2. Garrattograms after ciqc assessment and self-assessment of p53 IHC. Lab/ Core ciqc Assessment TP53 mutation status 1 A A A A N A A U U A U U A U U A A A U U A A A A A A U A A A A U A N A A U U Missense EC A A N A N A A U U A U N A N U A A A U U A U A A U A U A A A A U A N A A U U 1 2 U U U U U U N U U U U U U N U U U U U U U U A A U U U A U U N U U U U U U U NA MC U U U U U U N U U U U U U N U U U U U U U U A A U U U A U U N U U U U U U U 2 3 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N A N N Wild type EC N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N N N N A N N 3 4 N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N U N A N N NA CCC N N N A N N N N N N U N A N N N N N N A N A U U N N N N N N N N A U N A A N 4 5 N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NA CCC N N N A N N N N N N N N A N N N N N N A N N N N N N N N N N N N N N N N N N 5 6 A U A N A A N A A A A A A A A A A A U N A A N A N A A N U A A A A A A N A A Splice site HGSC A N A N A A N A A A A A N N A N N N A N A A N A N A A N U A N A N A N N N A 6 7 N N N N N N N N N N N N N N N N N N U N N N N N N N U N N N N A N U N N N N Wild type EC N N N N N N N N N N A A N N N N N N A N N N N N N N A N N N N A N A N N N N 7 8 A A A N A A N A A A A A A A A A A A U A A A A A N A A A A A A A A A A A A A Stopgain HGSC N A A N A A N A A A A A A A A A A A A N A A N A N A A A A A N A A A A A A A 8 9 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NA CCC N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N N N N N N N 9 10 N N N N N N N N N N U N N N N N N N U N N N N N N U U N N U N N N N N N N N NA CCC N N N N N N N N N N A A N N N N N N A N N N N A N A A N N A N N N N N N N N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A NA HGSC A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A N N N N N N N N N N N N N N N N N N A N N N N N N U N N N N N A N N N N N N NA LGSC N N N N N N N N N N N N N N N N N N A N N N N A N A N N N N N A N N N N N N A A A N A A N A A A A A A A A A A A A A A A A A A A A A A U A A A U A A A A Stopgain HGSC A A A N A A N A A A A A A A A A N A A N A A N U A A A A A N U A N U U A A A N N N N N N N N N N N N N N N N N N A N N N N N N A N N N N N A N N N N N N NA CCC N N N N N N N N N N N N N N N N N N A N N N N A N A N N N N N A N N N N N N A A A N A A N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Stopgain HGSC A A A N A A N A A A A A A A A A N A A N A A N A A A A A A A N A A A A A A A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N A N N N N N N NA CCC N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Wild type EC N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N U N N N N N N A N U N N N N N N N N N N A N N N N N N Wild type CCC N N N N N N N N N N A A N N N N N N A N A N N A N A N N N N N A N A N N N N N N N A N N N N N N N N N N N N N N A N N N N N A N N N N N N A N N N N N N Wild type EC N N N A N N N N N N N N A N N N N N A A N N N A A A N N N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NA CCC N N N N N N N N N N N N A A N N N N N A N N A N N U N N N N N N N N A N N N N U N A N N N N N N U N U N N N N N N N N N N N N U N N N N N N N N N U N N NA LGSC N U N A N N N N N N U N U N N N N N N A N N N N N U N N N N N N N N N U N N A A A N A A N A A A A A A A A A A A A N A A A A A A A A A A A A A A A A A A Stopgain HGSC A A A N A A N A A A A A A A A A A A A N A A A A A A A A A A N A A A A A A A A A A N A A N A A A A A A A A A A A A N A A A A A A A A A A A A A A A N A A Indel HGSC A A N N N A N N N A A N N N N N N N A N A N N A A A N A A A N A N A N N N N A A A N A A N A A A A A A A A A N A A A A A A A A A A A A A A A A A A A A A Indel HGSC A A A N A A N A A A A A A A A A N A A N A A A A A A N A A A N A A A A A A A N N N N N N N N N N N N N N N N N N A N N N N N N N N N N N N A N N N N N N Wild type EC N N N N N N N N N N N N A N N N N N A N N N N N N N N N N N N A N N N N N N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Missense HGSC A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NA CCC N N N N N N N N N N N N A N N N N N N A N N N N N N N N N N N N N N N N N N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Missense HGSC A A A A A A A A A A A A A A A A A A A A A A A A A A N A A A A A A A A A A A N U N N N N N N N N N N N N U N N N N N N N N N N N N N N N N N N N N N N N Wild type EC N U N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N N N N N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Missense HGSC A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A U U U A A A A A N A A A U N A A A N A N U A A A N A A A N A A A A A N A A A NA CCC U A A A A A A A N A A A A N A A A N A N U A A A N A A A N A A A A A N A A A A A A U A A N A A A A A A A A A A A A A A A U A A A A A A U U A A A A A A A Stopgain HGSC U A A U A A N A A A A A A A A A A A A N A A U A A A A A U U U A A A A A A A N N N N N N N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N NA MC N U N N N N N N N N U N N N N N N N A N N N N A N N N N N N N N N N N N N N U U U U U U N N U U U N N U U U N U U N U N N A U U A U U U N U N U U U N N Stopgain HGSC N U U U U U N N U U U N N U U U N U U N U N N A U U A U U U N U N U U U N N N N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NA CCC N N N A N N N N N N N N A N N N N N N A N N N N N N N N N N N N N N N N N N U U A U U U N U N U U A U N N U U U U N U U U A U U N N U U N N U U U U U N NA LGSC U U A U U U N U N U U A U N N U U U U N U U U A U U N N U U N N U U U U U N N N N N N N N N N N N N U N N N N N A N N N N N N N N N A N N N N A N N N N Missense HGSC N N N N N N N N N N N N A N N N N N A N N N N N N N N N A N N N N A N N N N N N N N N N N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N wild type CCC N N N N N N N N N N N N N N N N N N A N N N N N N N N N N N N N N N N N N N A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Missense HGSC A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A N A A A A N N N N N N N N N N N N N N N N N N A N N N N N N U N N N N N A N U N N N N Wild type EC N N N N N N N N N N N N N N N N N N A N N N N A N A N N N N N A N A N N N A N N N U N N N N N N N N N N N N N N N N N N N N N U N N N N N N N N N N N N Wild type EC N U N U N N N N N N U N A N N U N N N N N N N U N U N N N N N N N N N N N N A A A N A A N A A A A A A A A A A A A N A A A A N A A A A A A A A A A A A A Indel HGSC A A A N A A N A A A A A A A A A N A A N A A A A N A A A A A N A A A A A A A 42 Histotype Self-assessment N Normal A Abnormal ("all"/strong positive staining) A Abnormal ("nothing"/absent expression) N Normal A Abnormal ("all"/strong positive staining) U Uninterpretable (no internal control, tumor or core) Lab/ Core U Uninterpretable (no internal control) U Uninterpretable (no tumor or core)

4 Table S1. Reported p53 staining protocols. Lab ID Ag Retrieval Method Time for Ag Ab Supplier/ Time for Ab Amplificatio Enhancemen Ab Clone Ab Dilution Ab Lot No. Detection System Chromogen Retrieval (min) Vendor Incubation (min) n (Y/N) t (Y/N) 101 CC1 32 DO-7 1:800 DAKO OptiView N Y DAB 102 DAKO PT HIGH ph 10/20/10 DO7 Predilute DAKO RT DAKO ENVISION FLEX NO CUSO4 DAB+ 103 CC1 36 MINS Bp53-11 PRE Ventana G mins ULTRA VIEW DAB ultra view Y DAB 104 HIER 20 DO-7 RTU Dako polymer yes no DAB 106 Microwave pressure cooker 10 minutes DO-7 1:100 Biocare minutes MACH4 no no DAB 107 cc1 24 DO-7 1:100 Dako M Optiview DAB N Y DAB 110 DAKO PT High ph 9.0@97 C 20 min DO-7 1:800 DAKO min Dako Envision Flex N N DAB 111 CC1 48 MIN DO-7 1/900 CELL MARQUE D 32 MIN OPTIVIEW N Y DAB 112 BOND Epitope Retrieval 2 30 minutes DO-7 RTU LECIA minutes BOND Polyner Refine Detection no no DAB 114 CC1 32 DO-7 1/400 Dako Optiview N Y DAB 115 EnVision Flex TRS, High Ph 30 min DO-7 RTU Dako mins envision flex N N DAB 120 Waterbath 20 DO-7 pre-dilute Dako EnVision Flex Autostainer link48 n n DAB 124 CC1 32 min D0-7 Prà -diluã Ventana G Optiview o n dab 125 HIER-HIGH PH 30 D07 RTU Dako Polymer N N DAB Microwave Pressure Cooker, 35 Minutes Citrate Buffer, ph 6.0 DO-7 1:1000 Dako minutes Dako Envision Plus No No DAB Plus AUTOMATED (ULTRA CELL CONDITIONER 1) 52 MINUTES DO7 PREDILUTE VENTANA G MINUTES ULTRAVIEW DAB Y N DAB 132 high ph-dako Flex 20 DO-7 RTU Dako Dako Envison Flex n n DAB 133 HIER 64 minutes D07 predilute Roche G minutes polymer- Optiview n n dab 138 FLEX TRS High 20 (min) DO-7 RTU Dako (min) Envision FLEX+ N N FLEX DAB + sub-chromo 141 HIER 20 min D0-7 1:800 DAKO min Polymer no no DAB 144 CC1 16 min. Bp53-11 Pre-Dilute Ventana G min. Optiview No Yes, copper DAB 145 AUTOMATED HIER CC1 32 D07 1/30 CELL MARQUE VENTANA XT OPTIVIEW ihc v4 N N DAB 147 HIER ph9 20 DO-7 1:1500 DAKO Polymer (Leica Refine) n n DAB 148 CC1 64 MIN DO-7 RTU Ventana G MIN ULTRAVIEW DAB N N DAB 149 PT Link high ph 20 min at 97 C D07 RTU Dako IR EnVision Flex Yes No DAB 150 cc1 60 D0-7 r/u Roche E ultraview n cu DAB 155 CC1 30 Bp53-11 Prà -diluã Ventana E Ultraview Dab n n Dab 159 high ph 40 DO-7 pre-diluted Dako (Agilent) Flex N N DAB 160 CC1 64 MIN DO DAKO MIN ULTRA-VIEW N COPPER CUSO4 DAB 168 HIER 48 DO-7 RTU Dako Envision Flex + N N DAB 176 CC1 32 Bp Predilute Ventana F OptiView N N DAB 183 Ultra CC1 64 min p53(d07) predilute Roche F min Ultraview n n DAB 186 HIER 20 MIN DO-7 1:1000 DAKO MIN LEICA POLYMER N N DAB 202 HIER citrate ph 1 10 d0-7 RTU Leica Leica Refine detection kit no no DAB 209 HIER 20 mins at 97C and then cooling down to 85C for DO-7 Pre dilute Dako mins Envision plus N N DAB another 20mins 217 HIER CC1 64 DO-7 1:200 Dako Ultraview N Y DAB 228 HIER in EDTA bufferr 20 min DO7 1:2000 Dako min Bond refine detection system None None DAB 230 HIER 60 MINS ANTI-p53 NONE Ventana G mins iview N N DAB

5 Table S4. Descriptive statistics for p53 after ciqc assessment. Lab ID Total n % Pairwise complete Concordance with Cohen's Sensitivity Specificity scorable observations reference (%) kappa /38 (97%) /35 (97%) /39 (95%) /37 (59%) /39 (92%) /39 (95%) /42 (69%) /39 (92%) /39 (97%) /39 (95%) /36 (94%) /39 (90%) /37 (97%) /40 (95%) /38 (95%) /39 (95%) /40 (90%) /39 (97%) /34 (74%) /40 (85%) /37 (97%) /40 (93%) /40 (90%) /42 (93%) /39 (87%) /34 (91%) /38 (95%) /41 (93%) /38 (100%) /36 (94%) /41 (90%) /39 (74%) /40 (93%) /35 (94%) /39 (97%) /38 (84%) /39 (92%) /40 (93%)